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Te that the genome sizes differ drastically between the two morphologically indistinguishable Spironucleus species. There are also differences in codon usages and the occurrence of allelic sequence variation. A large number of S. barkhanus genes have high frequencies of SNPs, whereas S. salmonicida genes show sequence homogeneity. The presence and absence of SNPs among genes and genomes are the results of interplay of mutations and recombinatory events within and between isolates. In G. intestinalis, the presence of meiotic sex has been suggested [49], the nuclei have been suggested to meet in the cyst with the possibility of genetic exchange [22], and exchange of genetic material between natural isolates has been described [50]. Our data suggest that the outcome of these different putative processes may be rather different in closely related diplomonads. Further studies are obviously needed to understand the importance of the various putative phenomena acting on diplomonad genomes. The observations in this study are in agreement with our studies of G. intestinalis genomes [7]. Thus, large genomic differences between morphologically indistinguishable isolates are widespread among diplomonads. MethodsOrganisms and culturesTrophozoites of S. barkhanus were isolated from the gall bladder of the freshwater salmonid grayling in Glomma River, south-eastern Norway. An axenic culture of S. barkhanus was grown at 4 in TYI-S-33 medium supplemented with bile according to Keister [51]. The identity of the isolate was verified by sequencing of the small subunit ribosomal RNA gene PCR amplified from the genome [52]. S. salmonicida (ATCC 50377), isolated fromRoxstr -Lindquist et al. BMC Genomics 2010, 11:258 http://www.biomedcentral.com/1471-2164/11/Page 10 ofANumber of cells (normalized to highest peak) Number of cells (normalized to highest peak)CB4N8N4N8NDDNA contentDNA contentFigure 3 Flow cytometry analysis of genomic size of S. barkhanus and S. salmonicida. (A) and (C) Flow-cytometric analysis of the total DNA content of exponentially growing G. intestinalis WB trophozoites. The G1 and G2/M peaks are labeled with 4N and 8N, corresponding to a total genome content of 48 and 96 Mbp. (B) Flow-cytometric analysis of the total DNA content of exponentially growing S. barkhanus trophozoites. (D) Flow-cytometric analysis of the total DNA content of exponentially growing S. salmonicida trophozoites.a muscle abscess in Atlantic salmon Vesteraalen, northern Norway (previously known as S. barkhanus [11]) was obtained from American Type CulCapivasertibCapivasertib Abstract(s)">PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 protocol.RNA isolation, cDNA library construction and sequencingSequencing Kit for MegaBACE DNA analysis systems (Amersham Biosciences).Sequence analysesExponentially growing S. barkhanus were harvested at passage 13. After collection by centrifugation (5 min at 2000 rpm, 4 ) the cells were directly lysed in Trizol reagent (Invitrogen) and total RNA was isolated according to the manufacturer's instruction. The amount and quality were analyzed by nanodrop and formaldehydedenaturing 1 agarose gel before total RNA was ethanol precipitated. Following the isolation of mRNA using Poly(A)PuristTM MAG system (Ambion), directed and size-fractionated cDNA libraries was made using the CloneMinerTM cDNA Library Construction Kit (Invitrogen). Positive transformants were manually picked for sequencing. Sequencing template preparation was made with the TempliPhi DNA.